Introduction: Little is known about the characteristics of the CD4+ T cells residing in close proximity to the tumor cells in classical Hodgkin lymphoma (cHL). We recently established that these CD4+ T cells interact with tumor cells by binding of CD2 to CD58 and T cell receptor to HLA class II in vitro and in situ. Formation of this immunological synapse results in strong T cell activation under normal conditions, but the CD4+ T cells in cHL are only weakly activated and typically lack expression of the activation marker CD26. The aim of this study was to further characterize these T cells.
Methods: CD4+CD26- and CD4+CD26+ T cell subsets were sorted from 19 cHL lymph node derived cell suspensions (tumor cells HLA class II positive) and analyzed by RNA sequencing and T cell receptor variable gene segment usage. In addition, co-expression of genes of interest was investigated at the single cell level using previously generated single-cell RNA sequencing (scRNA-seq) data and at the protein level by immunohistochemistry.
Results: Gene set variation analysis showed an enrichment of memory Treg, Th17 and T follicular helper cell gene signatures in CD4+CD26- T cells, while naïve and Th1/17 gene cell signatures were enriched in CD4+CD26+ T cells. Although CD4+CD26- T cells displayed an antigen experienced phenotype, the T cell receptor variable gene segment usage was polyclonal and did not differ from CD4+CD26+ T cells, indicating lack of clonal expansion. Differential gene expression analysis revealed a significant enrichment of 100 genes in CD4+CD26- T cells. Seven genes (TOX, TOX2, CXCL13, CTTN, PDCD1, CD200 and NFIA) with a moderate to high expression level were chosen for subsequent co-expression analysis using scRNA-seq data. This revealed that the majority of CD4+CD26- T cells expressed TOX2 either alone or in combination with any of the other selected genes. Protein expression of TOX and TOX2, transcription factors crucial for the acquisition of an exhausted T cell phenotype, was accentuated in T cells that physically interact with tumor cells. More than 50% of these rosetting T cells were positive for TOX in 63% and TOX2 in 79% of cHL cases.
Conclusion: CD4+ T cells residing in close proximity to cHL tumor cells are polyclonal, antigen experienced and exhausted. We propose that TOX and TOX2, transcription factors known to induce expression of a variety of immune checkpoints, are attractive therapeutic targets for cHL.